Antibody-based screening of cell wall matrix glycans in ferns reveals taxon, tissue and cell-type specific distribution patterns Leroux et al. Leroux et al. BMC Plant Biology (2015) 15:56 DOI 10.1186/s12870-014-0362-8 Leroux et al. BMC Plant Biology (2015) 15:56 DOI 10.1186/s12870-014-0362-8 RESEARCH ARTICLE Open Access Antibody-based screening of cell wall matrix glycans in ferns reveals taxon, tissue and cell-type specific distribution patterns Olivier Leroux1*, Iben Sørensen2,3, Susan E Marcus4, Ronnie LL Viane1, William GT Willats2 and J Paul Knox4 Abstract Background: While it is kno3wn that complex tissues with specialized functions emerged during land plant evolution, it is not clear how cell wall polymers and their structural variants are associated with specific tissues or cell types. Moreover, due to the economic importance of many flowering plants, ferns have been largely neglected in cell wall comparative studies. Results: To explore fern cell wall diversity sets of monoclonal antibodies directed to matrix glycans of angiosperm cell walls have been used in glycan microarray and in situ analyses with 76 fern species and four species of lycophytes. All major matrix glycans were present as indicated by epitope detection with some variations in abundance. Pectic HG epitopes were of low abundance in lycophytes and the CCRC-M1 fucosylated xyloglucan epitope was largely absent from the Aspleniaceae. The LM15 XXXG epitope was detected widely across the ferns and specifically associated with phloem cell walls and similarly the LM11 xylan epitope was associated with xylem cell walls. The LM5 galactan and LM6 arabinan epitopes, linked to pectic supramolecules in angiosperms, were associated with vascular structures with only limited detection in ground tissues. Mannan epitopes were found to be associated with the development of mechanical tissues. We provided the first evidence for the presence of MLG in leptosporangiate ferns. Conclusions: The data sets indicate that cell wall diversity in land plants is multifaceted and that matrix glycan epitopes display complex spatio-temporal and phylogenetic distribution patterns that are likely to relate to the evolution of land plant body plans. Keywords: Cell wall evolution, Homogalacturonan, Arabinan, Galactan, Xyloglucan, Xylan, Mannan, Mixed-linkage glucan, Sclerenchyma Background and cell types, especially in the vegetative body, emerged The colonisation of land was a major event in the history and contributed to the structural complexity of plants. As of plants. Subsequent widespread ecological radiation and the architecture and properties of cell walls largely deter- diversification was directed by complex interactions involv- mine tissue/organ structure and function and consequently ing the interplay between morpho-anatomical and physio- overall morphology, they must have played a fundamental logical adaptations of plants and the physical and chemical role in the evolution and differentiation of complex body changes in their environment. Many adaptations facilitated plans. terrestrial colonisation and survival, including anchorage By the end of the 19th century, the combined efforts of and water uptake, mechanical support, water transport, many plant anatomists led to an increased knowledge of protection against desiccation and UV-irradiance, as well the anatomical complexity of land plants, resulting in as reproduction in absence of water [1]. Specialised tissues the distinction of tissues and cell types that are still recognised today [2]. These tissues are composed of cells with walls that are classed as either primary cell walls * Correspondence: [email protected] that prevent cell bursting and regulate cell expansion, or 1Pteridology, Department of Biology, Ghent University, K.L. Ledeganckstraat 35, Ghent B-9000, Belgium non-extendable secondary cell walls, restricted to certain Full list of author information is available at the end of the article cell types, which have mechanical properties resisting © 2015 Leroux et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Leroux et al. BMC Plant Biology (2015) 15:56 Page 2 of 19 external forces that would lead to cell collapse. Both [19,20] has indicated structurally distinct cell walls that do types of walls are structurally complex composites. In not fit within either the type I or type II classification that most primary cell walls a load bearing network of cellu- had been developed for angiosperm cell walls [21,22]. Re- lose microfibrils is cross-linked and interspersed with cently, a third mannan-rich (primary) cell wall type (cell complex sets of matrix glycans including those classed wall type III), typical of ferns was reported [23]. Although as hemicelluloses (xyloglucans, heteroxylans, heteroman- broadly useful in reflecting major taxonomic distinctions nans and mixed-linkage glucans) and the multi-domain in global compositional differences, classifications of cell pectic supramolecular polysaccharides [3,4]. Secondary wall types neglects variation in wall components between cell walls are often reinforced with lignin and contain cell types within organs and most notably may not relate low amounts of pectins. Many cell wall components may to all land plant species. In addition, little is known of how display considerable heterogeneity, either in their mo- the range of polysaccharides found in primary and second- lecular structure or in their spatio-temporal distribution ary cell walls relates to the evolution of specific cell wall in plant organs, tissues, cell-types and individual walls functions and cell types. [3,5]. As wall components may be present in variable To develop a deeper understanding of cell wall diver- amounts in different cell walls at specific developmental sity within the context of tissues, cell types and individ- stages, there is not always a clear distinction in molecu- ual walls in a group of land plants that has not been lar composition between primary and secondary cell previously extensively studied, we carried out a glycan walls [6]. Moreover, walls may be modified in response microarray analysis complemented with selected in situ to environmental stress or pathogen attack [7] and even immunolabelling of 76 fern species and 4 lycophytes after cell death (e.g. postmortem lignification [8]). species (Figure 1). Through extensive sampling within Cell walls also display remarkable diversity at the taxo- leptosporangiate ferns, and Aspleniaceae in particular, nomical level as the presence and/or abundance of spe- we aimed to identify tissue or cell type-specific distribu- cific wall components may vary between the major plant tion patterns of matrix glycan epitopes, but also explore lineages (e.g. [9-17]; see [18] for a brief overview). Analysis variation in matrix glycan cell wall composition at family of the early diverging fern (s.l., monilophyta) Equisetum and species levels. Figure 1 Schematic tree showing the relationships among the major groups of land plants. 1: eusporangiate ferns s.l.; 2: homosporous lycophytes; 3: heterosporous lycophytes. Representatives of the plant groups indicated in bold were sampled for this study (see Supplementary Figure 1). Genera represented in the immunofluorescence figures are indicated (grey). Adapted from [74,75]. Leroux et al. BMC Plant Biology (2015) 15:56 Page 3 of 19 Results and discussion not sample all organs and structures (including roots, Interpretation of the glycan microarray analysis was rhizomes and laminae but also meristems and differenti- approached from the perspective of cell wall polysac- ating tissues) for each of the species studied, we can by charide classes and the results are presented as heatmaps no means state that certain epitopes are absent in the (Figures 2, 3 and 4). An exploratory glycan microarray plant. analysis of organs and tissues of the leptosporangiate To understand the variation in epitope abundance we fern Asplenium elliotti revealed considerable variation performed in situ immunolabelling experiments using in the relative abundance of glycan epitopes among sam- the same antibodies as used for probing the glycan mi- ples with most epitopes being detected in the petiole tis- croarrays. As mAbs are epitope-specific and not polymer- sues (Figure 2). As our aim was to explore tissue-specific specific, and, some epitopes might be masked by other distribution of glycan epitopes across ferns we performed a wall components [24], we cannot draw any firm conclu- broad-scale glycan microarray analysis by sampling only sions on general fern cell wall composition. However, im- petiole bases (or stems in the case of Huperzia, Selaginella, munofluorescence (IF) is a powerful tool to explore spatial Psilotum and Equisetum). The resulting heatmaps are patterns in glycan-epitope distribution, which is the main shown in relation to both fern division and molecular aim of this study. probe class (Figures 3 and 4). Broad themes that became apparent in the glycan epi- Variation in the dataset may reflect differences in tope analysis included the observation that the majority developmental stage